Gregorio Valdez

MicroRNA-206 Delays ALS Progression and Promotes Regeneration of Neuromuscular Synapses in Mice

An Innervative Small RNA Amyotrophic lateral sclerosis (ALS) is a relentless disease characterized by progressive degeneration of motor neurons that control muscle movement, leading to muscle atrophy and paralysis. Williams et al. (p. 1549; see the Perspective by Brown) show that a small noncoding RNA that is selectively expressed in skeletal muscle, miR-206, senses motor neuron injury or loss and helps ameliorate resultant muscle damage by promoting regeneration of neuromuscular synapses. Expression of miR-206 was dramatically induced in a mouse model of ALS, and when this RNA was removed from mice by genetic manipulation, the disease progressed at a faster rate. The salutary effects of miR-206 appear to be mediated through a signaling pathway in muscle cells involving histone deacetylase 4 and a fibro-blast growth factor modulator, activation of which leads to release of factors that promote nerve-muscle interactions. A small noncoding RNA promotes nerve-muscle interactions in response to motor neuron injury and slows disease progression. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by loss of motor neurons, denervation of target muscles, muscle atrophy, and paralysis. Understanding ALS pathogenesis may require a fuller understanding of the bidirectional signaling between motor neurons and skeletal muscle fibers at neuromuscular synapses. Here, we show that a key regulator of this signaling is miR-206, a skeletal muscle–specific microRNA that is dramatically induced in a mouse model of ALS. Mice that are genetically deficient in miR-206 form normal neuromuscular synapses during development, but deficiency of miR-206 in the ALS mouse model accelerates disease progression. miR-206 is required for efficient regeneration of neuromuscular synapses after acute nerve injury, which probably accounts for its salutary effects in ALS. miR-206 mediates these effects at least in part through histone deacetylase 4 and fibroblast growth factor signaling pathways. Thus, miR-206 slows ALS progression by sensing motor neuron injury and promoting the compensatory regeneration of neuromuscular synapses.

A Neurotrophin Signaling Cascade Coordinates Sympathetic Neuron Development through Differential Control of TrkA Trafficking and Retrograde Signaling

A fundamental question in developmental biology is how a limited number of growth factors and their cognate receptors coordinate the formation of tissues and organs endowed with enormous morphological complexity. We report that the related neurotrophins NGF and NT-3, acting through a common receptor, TrkA, are required for sequential stages of sympathetic axon growth and, thus, innervation of target fields. Yet, while NGF supports TrkA internalization and retrograde signaling from distal axons to cell bodies to promote neuronal survival, NT-3 cannot. Interestingly, final target-derived NGF promotes expression of the p75 neurotrophin receptor, in turn causing a reduction in the sensitivity of axons to intermediate target-derived NT-3. We propose that a hierarchical neurotrophin signaling cascade coordinates sequential stages of sympathetic axon growth, innervation of targets, and survival in a manner dependent on the differential control of TrkA internalization, trafficking, and retrograde axonal signaling.

Attenuation of age-related changes in mouse neuromuscular synapses by caloric restriction and exercise

The cellular basis of age-related behavioral decline remains obscure but alterations in synapses are likely candidates. Accordingly, the beneficial effects on neural function of caloric restriction and exercise, which are among the most effective anti-aging treatments known, might also be mediated by synapses. As a starting point in testing these ideas, we studied the skeletal neuromuscular junction (NMJ), a large, accessible peripheral synapse. Comparison of NMJs in young adult and aged mice revealed a variety of age-related structural alterations, including axonal swellings, sprouting, synaptic detachment, partial or complete withdrawal of axons from some postsynaptic sites, and fragmentation of the postsynaptic specialization. Alterations were significant by 18 mo of age and severe by 24 mo. A life-long calorie-restricted diet significantly decreased the incidence of pre- and postsynaptic abnormalities in 24-mo-old mice and attenuated age-related loss of motor neurons and turnover of muscle fibers. One month of exercise (wheel running) in 22-mo-old mice also reduced age-related synaptic changes but had no effect on motor neuron number or muscle fiber turnover. Time-lapse imaging in vivo revealed that exercise partially reversed synaptic alterations that had already occurred. These results demonstrate a critical effect of aging on synaptic structure and provide evidence that interventions capable of extending health span and lifespan can partially reverse these age-related synaptic changes.

Pincher, a pinocytic chaperone for nerve growth factor/TrkA signaling endosomes

Acentral tenet of nerve growth factor (NGF) action that is poorly understood is its ability to mediate cytoplasmic signaling, through its receptor TrkA, that is initiated at the nerve terminal and conveyed to the soma. We identified an NGF-induced protein that we termed Pincher (pinocytic chaperone) that mediates endocytosis and trafficking of NGF and its receptor TrkA. In PC12 cells, overexpression of Pincher dramatically stimulated NGF-induced endocytosis of TrkA, unexpectedly at sites of clathrin-independent macropinocytosis within cell surface ruffles. Subsequently, a system of Pincher-containing tubules mediated the delivery of NGF/TrkA-containing vesicles to cytoplasmic accumulations. These vesicles selectively and persistently mediated TrkA-erk5 mitogen-activated protein kinase signaling. A dominant inhibitory mutant form of Pincher inhibited the NGF-induced endocytosis of TrkA, and selectively blocked TrkA-mediated cytoplasmic signaling of erk5, but not erk1/2, kinases. Our results indicate that Pincher mediates pinocytic endocytosis of functionally specialized NGF/TrkA endosomes with persistent signaling potential.

Pincher-Mediated Macroendocytosis Underlies Retrograde Signaling by Neurotrophin Receptors

Retrograde signaling by neurotrophins is crucial for regulating neuronal phenotype and survival. The mechanism responsible for retrograde signaling has been elusive, because the molecular entities that propagate Trk receptor tyrosine kinase signals from the nerve terminal to the soma have not been defined. Here, we show that the membrane trafficking protein Pincher defines the primary pathway responsible for neurotrophin retrograde signaling in neurons. By both immunofluorescence confocal and immunoelectron microscopy, we find that Pincher mediates the formation of newly identified clathrin-independent macroendosomes for Trk receptors in soma, axons, and dendrites. Trk macroendosomes are derived from plasma membrane ruffles and subsequently processed to multivesicular bodies. Pincher similarly mediates macroendocytosis for NGF (TrkA) and BDNF (TrkB) in both peripheral (sympathetic) and central (hippocampal) neurons. A unique feature of Pincher-Trk endosomes is refractoriness to lysosomal degradation, which ensures persistent signaling through a critical effector of retrograde survival signaling, Erk5 (extracellular signal-regulated kinase 5). Using sympathetic neurons grown in chamber cultures, we find that block of Pincher function, which prevents Trk macroendosome formation, eliminates retrogradely signaled neuronal survival. Pincher is the first distinguishing molecular component of a novel mechanistic pathway for endosomal signaling in neurons.

Shared Resistance to Aging and ALS in Neuromuscular Junctions of Specific Muscles

Normal aging and neurodegenerative diseases both lead to structural and functional alterations in synapses. Comparison of synapses that are generally similar but respond differently to insults could provide the basis for discovering mechanisms that underlie susceptibility or resistance to damage. Here, we analyzed skeletal neuromuscular junctions (NMJs) in 16 mouse muscles to seek such differences. We find that muscles respond in one of three ways to aging. In some, including most limb and trunk muscles, age-related alterations to NMJs are progressive and extensive during the second postnatal year. NMJs in other muscles, such as extraocular muscles, are strikingly resistant to change. A third set of muscles, including several muscles of facial expression and the external anal sphinter, succumb to aging but not until the third postnatal year. We asked whether susceptible and resistant muscles differed in rostrocaudal or proximodistal position, source of innervation, motor unit size, or fiber type composition. Of these factors, muscle innervation by brainstem motor neurons correlated best with resistance to age-related decline. Finally, we compared synaptic alterations in normally aging muscles to those in a mouse model of amyotrophic lateral sclerosis (ALS). Patterns of resistance and susceptibility were strikingly correlated in the two conditions. Moreover, damage to NMJs in aged muscles correlated with altered expression and distribution of CRMP4a and TDP-43, which are both altered in motor neurons affected by ALS. Together, these results reveal novel structural, regional and molecular parallels between aging and ALS.

Trk-signaling endosomes are generated by Rac-dependent macroendocytosis

Why neurotrophins and their Trk receptors promote neuronal differentiation and survival whereas receptor tyrosine kinases for other growth factors, such as EGF, do not, has been a long-standing question in neurobiology. We provide evidence that one difference lies in the selective ability of Trk to generate long-lived signaling endosomes. We show that Trk endocytosis is distinguished from the classical clathrin-based endocytosis of EGF receptor (EGFR). Although Trk and EGFR each stimulate membrane ruffling, only Trk undergoes both selective and specific macroendocytosis at ruffles, which uniquely requires the Rho-GTPase, Rac, and the trafficking protein, Pincher. This process leads to Trk-signaling endosomes, which are immature multivesicular bodies that retain Rab5. In contrast, EGFR endosomes rapidly exchange Rab5 for Rab7, thereby transiting into late-endosomes/lysosomes for degradation. Sustained endosomal signaling by Trk does not reflect intrinsic differences between Trk and EGFR, because each elicits long-term Erk-kinase activation from the cell surface. Thus, a population of stable Trk endosomes, formed by specialized macroendocytosis in neurons, provides a privileged endosome-based system for propagation of signals to the nucleus.

Laminins promote postsynaptic maturation by an autocrine mechanism at the neuromuscular junction.

A prominent feature of synaptic maturation at the neuromuscular junction (NMJ) is the topological transformation of the acetylcholine receptor (AChR)-rich postsynaptic membrane from an ovoid plaque into a complex array of branches. We show here that laminins play an autocrine role in promoting this transformation. Laminins containing the α4, α5, and β2 subunits are synthesized by muscle fibers and concentrated in the small portion of the basal lamina that passes through the synaptic cleft at the NMJ. Topological maturation of AChR clusters was delayed in targeted mutant mice lacking laminin α5 and arrested in mutants lacking both α4 and α5. Analysis of chimeric laminins in vivo and of mutant myotubes cultured aneurally demonstrated that the laminins act directly on muscle cells to promote postsynaptic maturation. Immunohistochemical studies in vivo and in vitro along with analysis of targeted mutants provide evidence that laminin-dependent aggregation of dystroglycan in the postsynaptic membrane is a key step in synaptic maturation. Another synaptically concentrated laminin receptor, Bcam, is dispensable. Together with previous studies implicating laminins as organizers of presynaptic differentiation, these results show that laminins coordinate post- with presynaptic maturation.

The mammalian exocyst, a complex required for exocytosis, inhibits tubulin polymerization

The exocyst is a 734-kDa complex essential for development. Perturbation of its function results in early embryonic lethality. Extensive investigation has revealed that this complex participates in multiple biological processes, including protein synthesis and vesicle/protein targeting to the plasma membrane. In this article we report that the exocyst may also play a role in modulating microtubule dynamics. Using monoclonal antibodies, we observed that endogenous exocyst subunits co-localized with microtubules and mitotic spindles in normal rat kidney cells. To test for a functional relationship between the exocyst complex and microtubules, we established an in vitro exocyst reconstitution assay and studied exocyst effect on microtubule dynamics. We found that the exocyst complex reconstituted from eight recombinant exocyst subunits inhibited tubulin polymerization in vitro. Deletion of exocyst subunit sec5, sec6, sec15, or exo70 diminished its tubulin polymerization inhibition activity. Surprisingly, exocyst subunit exo70 itself was also capable of inhibiting tubulin polymerization, although exocyst complex with exo70 deletion did not lose its activity completely. Overexpression of exo70 in NRK cells resulted in microtubule network disruption and the formation of filopodia-like plasma membrane protrusions. The formation of these membrane protrusions was greatly hampered by stabilizing microtubules with taxol. Overexpression of exo84, an exocyst subunit that did not show tubulin polymerization inhibition activity, did not cause this phenotype. Results shown in this article, along with a previous report that localized microtubule instability induces plasma membrane addition, implicates a novel role for the exocyst in modulating microtubule dynamics underlying exocytosis.

Agrin and synaptic laminin are required to maintain adult neuromuscular junctions.

As synapses form and mature the synaptic partners produce organizing molecules that regulate each other’s differentiation and ensure precise apposition of pre- and post-synaptic specializations. At the skeletal neuromuscular junction (NMJ), these molecules include agrin, a nerve-derived organizer of postsynaptic differentiation, and synaptic laminins, muscle-derived organizers of presynaptic differentiation. Both become concentrated in the synaptic cleft as the NMJ develops and are retained in adulthood. Here, we used mutant mice to ask whether these organizers are also required for synaptic maintenance. Deletion of agrin from a subset of adult motor neurons resulted in the loss of acetylcholine receptors and other components of the postsynaptic apparatus and synaptic cleft. Nerve terminals also atrophied and eventually withdrew from muscle fibers. On the other hand, mice lacking the presynaptic organizer laminin-α4 retained most of the synaptic cleft components but exhibited synaptic alterations reminiscent of those observed in aged animals. Although we detected no marked decrease in laminin or agrin levels at aged NMJs, we observed alterations in the distribution and organization of these synaptic cleft components suggesting that such changes could contribute to age-related synaptic disassembly. Together, these results demonstrate that pre- and post-synaptic organizers actively function to maintain the structure and function of adult NMJs.